Pertussis vaccines and methods of making and using

ABSTRACT

Bordetella pertussis  iron receptor proteins or portions thereof (e.g., one or more extracellular domains), alone or spliced into  B. pertussis  scaffold proteins (e.g., fimbrial or flagellin), are provided and can be used in acellular vaccines to protect against pertussis or other  Bordetella  diseases in humans and non-human mammals. In addition,  Bordetella  species grown under iron-starved conditions are provided and can be used in whole cell vaccines to protect against pertussis or other  Bordetella  diseases in humans and non-human mammals.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority under 35 U.S.C. 119(e)to U.S. Application No. 62/315,356 filed Mar. 30, 2016.

FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under AI31088 awarded bythe National Institutes of Health. The government has certain rights inthe invention.

TECHNICAL FIELD

This disclosure generally relates to vaccines.

BACKGROUND

Bordetella pertussis is the bacterial agent of whooping cough(pertussis). The primary pertussis vaccines currently in use in the U.S.are acellular (e.g., INFANRIX and BOOSTRIX (GlaxoSmithKline); DAPTACELand ADACEL (Sanofi Pasteur) [infant and adult formulations for each,respectively]). The pertussis component of these vaccines containsdetoxified pertussis toxin and two surface proteins (e.g., filamentoushemagglutinin, pertactin), while DAPTACEL and ADACEL also containfimbrial (Fim) proteins, Fim2 and Fim3. For a number of differentreasons (e.g., current acellular pertussis vaccines do not providedurable immunity; current acellular pertussis vaccines prevent diseasesymptoms but do not prevent B. pertussis colonization; and/or B.pertussis strains have been isolated from patients that lack aparticular protein component of the vaccine), these acellular vaccineshave been ineffective in preventing colonization and transmission of B.pertussis. Therefore, a new approach to impart immunity against B.pertussis is needed.

SUMMARY

Currently used acellular pertussis vaccines typically include a smallset of antigens representing three or four virulence factors from B.pertussis. It is now apparent from the significant increase in pertussiscases and from epidemiological studies that these acellular vaccineslack the desired level of efficacy and duration of protection.

This disclosure provides a solution to this problem by describingpolypeptides that can be used as vaccines to inoculate against one ormore Bordetella species (e.g., B. pertussis, B. parapertussis, B.bronchiseptica, and/or B. avium). The vaccines described herein providesignificantly improved immunity against B. pertussis over existingacellular pertussis vaccines. Further, a similar strategy can be appliedto provide a whole-cell pertussis vaccine that imparts improved immunityrelative to the whole-cell pertussis vaccines that are used in manycountries throughout the world.

In one aspect, a chimeric polypeptide is provided that includes at leastone antigenic polypeptide and a scaffold protein.

In some embodiments, the at least one antigenic polypeptide includes aniron receptor protein or an antigenic portion thereof. In someembodiments, the antigenic portion of an iron receptor protein includesat least one extracellular domain. In some embodiments, the ironreceptor protein is a TonB-dependent receptor protein or an antigenicportion thereof. A representative TonB-dependent receptor protein is aferric enterobactin siderophore (BfeA) receptor protein. In someembodiments, the iron receptor protein is a hemin or hemoproteinreceptor or an antigenic portion thereof. A representative hemin orhemoprotein receptor is a BhuR protein. In some embodiments, the ironreceptor protein is a siderophore receptor or an antigenic portionthereof. A representative siderophore receptor is an alcaliginsiderophore receptor (FauA).

In some embodiments, the scaffold protein is a fimbrial protein or aflagellin protein. Representative fimbrial proteins include, withoutlimitation, a fimbrial 2 protein or fimbrial 3 protein. A representativeflagellin protein is a flagellin subunit protein.

In one aspect, a nucleic acid molecule encoding a polypeptide asdescribed herein (e.g., a chimeric polypeptide) is provided. In anotheraspect, a construct that includes such a nucleic acid molecule isprovided. In still another aspect, a host cell that includes such anucleic acid molecule or construct is provided.

In another aspect, an acellular B. pertussis vaccine for protecting asubject against infection by B. pertussis, B. parapertussis, B.bronchiseptica and/or B. avium is provided. Such a vaccine typicallyincludes a chimeric polypeptide as described herein and apharmaceutically acceptable carrier. In some embodiments, such anacellular vaccine further includes an adjuvant.

In still another aspect, a whole cell B. pertussis vaccine forprotecting a subject against infection by B. pertussis, B.parapertussis, B. bronchiseptica and/or B. avium is provided. Such avaccine typically includes a composition of B. pertussis grown underiron-starvation conditions and a pharmaceutically acceptable carrier. Insome embodiments, such a whole cell vaccine further includes anadjuvant.

In yet another aspect, a method of vaccinating a subject against B.pertussis is provided. Such a method typically includes administering apolypeptide as described herein (e.g., a chimeric polypeptide) or avaccine as described herein to a subject. Representative subjectsinclude, without limitation, humans, canines, pigs, rabbits, cats, orbirds.

In still another aspect, a method of making an acellular B. pertussisvaccine is provided. Such a method typically includes providing apolypeptide as described herein (e.g., a chimeric polypeptide); orexpressing a nucleic acid molecule or construct as described herein; orculturing a host cell as described herein (e.g., a host cell expressinga nucleic acid molecule or construct as described herein); and combiningthe polypeptide produced therefrom with a pharmaceutically acceptablecarrier. In some embodiments, such a method further includes adding anadjuvant.

In yet another aspect, a method of making a whole-cell B. pertussisvaccine is provided. Such a method typically includes culturing B.pertussis under iron-starvation conditions; and processing the B.pertussis grown under iron-starvation conditions into a whole-cellvaccine. In some embodiments, such a method further includes adding anadjuvant.

In one aspect, a polypeptide that includes at least one extracellulardomain of an iron receptor protein is provided. In some embodiments, theiron receptor protein is a heme, hemin or hemoprotein receptor (e.g., aBhuR protein). In some embodiments, the iron receptor protein is asiderophore receptor (e.g., an alcaligin siderophore receptor [FauA]).In some embodiments, the iron receptor protein is a TonB-dependentreceptor protein (e.g., an enterobactin siderophore receptor protein[BfeA]).

In another aspect, a chimeric polypeptide that includes a scaffoldprotein or portion thereof and at least one antigenic polypeptide isprovided. In some embodiments, the at least one antigenic polypeptide isspliced into a scaffold protein. Representative scaffold proteinsinclude a fimbrial 2 or a fimbrial 3 protein or a flagellin protein. Insome embodiments, the at least one antigenic polypeptide includes atleast one extracellular domain of an iron receptor protein. In someembodiments, the at least one antigenic polypeptide includes an ironreceptor protein. In some embodiments, the at least one antigenicpolypeptide includes at least one extracellular domain of aTonB-dependent receptor protein. In some embodiments, the at least oneantigenic polypeptide includes a TonB-dependent receptor protein.

In still another aspect, a nucleic acid molecule is provided thatencodes any of the polypeptides described herein. In yet another aspect,a construct is provided that includes a nucleic acid molecule asdescribed herein. In another aspect, a host cell is provided thatincludes a nucleic acid molecule as described herein or a construct asdescribed herein.

In one aspect, an acellular B. pertussis vaccine is provided forprotecting a subject against infection by B. pertussis, B.parapertussis, B. bronchiseptica and/or B. avium. Such a vaccinetypically includes any of the polypeptides described herein. In anotheraspect, a whole cell B. pertussis vaccine is provided for protecting asubject against infection by B. pertussis, B. parapertussis, B.bronchiseptica and/or B. avium. Such a vaccine typically includes B.pertussis grown under iron-starved conditions that promote theproduction of iron receptors. In some embodiments, a vaccine further caninclude an adjuvant.

In still another aspect, a method of vaccinating a subject against B.pertussis is provided. Such a method typically includes administering,to a subject, any of the polypeptides described herein, any of thenucleic acid molecules described herein, any of the constructs describedherein, any of the host cells described herein, or any of the vaccinesdescribed herein. In some embodiments, the subject is a human, a canine,a pig, a rabbit, a cat, or a bird.

In still another aspect, a method of making an acellular B. pertussisvaccine is provided. Such a method typically includes providing any ofthe polypeptides described herein; or expressing any of the nucleic acidmolecules described herein; or expressing any of the constructsdescribed herein; or culturing any of the host cells described herein;and combining the polypeptide produced therefrom with a pharmaceuticallyacceptable carrier. In yet another aspect, a method of making awhole-cell B. pertussis vaccine is provided. Such a method typicallyincludes culturing B. pertussis under iron-starvation conditions thatenhance iron receptor production; and processing the iron-starved B.pertussis cells for delivery as a vaccine. In some embodiments, such amethod further includes adding an adjuvant.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the methods and compositions of matter belong. Althoughmethods and materials similar or equivalent to those described hereincan be used in the practice or testing of the methods and compositionsof matter, suitable methods and materials are described below. Inaddition, the materials, methods, and examples are illustrative only andnot intended to be limiting. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety.

DESCRIPTION OF DRAWINGS

FIG. 1A is a photograph of an immunoblot showing total membranesproteins prepared from B. pertussis (Bp) and B. bronchiseptica (Bb)cultured under iron-replete (+Fe) and iron-depleted (−Fe) conditionsprobed using human sera from normal uninfected donors and from infectedpertussis patients.

FIG. 1B is a photograph showing the juxtaposition of B. pertussis FIG.1A pertussis patient immunoblot lanes; immunoreactive iron-regulatedproteins are indicated with small triangles.

FIG. 1C is a photograph showing total membrane proteins of B. pertussisand B. bronchiseptica, and outer membrane proteins from B.bronchiseptica strains that overproduce the FauA, BhuR or BfeA receptorsof B. pertussis, probed using sera from normal and culture-positivedonors (different donors than in FIG. 1A).

FIG. 2 is a graph showing that B. pertussis mutants lacking the FauA andBhuR receptors are attenuated in competition infections using a mousemodel of pertussis. Mice (5/group) were co-infected with a 1:1 mixtureof wild type B. pertussis and a receptor mutant derivative. Bacterialcolony forming units (CFU) of each strain pair in the lungs and tracheawere determined at different times post-infection. The competitive indexis the ratio of the mutant/wild-type CFU ratio recovered at each timepoint divided by the mutant/wild-type CFU ratio in the input inoculum(mean values shown). A competitive index of <1 indicates the mutantstrain is less fit (exhibits less growth in mice) compared with the wildtype parent strain. A plot (grey line) showing CFU levels typical of B.pertussis mouse respiratory tract infections is overlaid for reference.A B. pertussis fauA mutant is defective for growth in the mousethroughout infection and a bhuR mutant is most defective during lateinfection. A double mutant lacking both fauA and bhuR is avirulent

FIG. 3 is a schematic showing the structural domains of the B. pertussisFauA and BhuR receptor proteins, including the cell surface-exposedloops that connect the antiparallel transmembrane beta strands (wideyellow ribbons). Both proteins have 11 flexible extracellular loops(within dashed boxes in structures, and dark regions within “Barrel”region). BhuR has an additional N-terminal extension (NTE) involved incell surface signaling. A linear depiction of the loop domain locationsis shown below the structures.

FIG. 4A shows a photograph of a SDS-PAGE gel showing purification ofpolyHis-tagged Bp FauA protein produced in Escherichia coli. M,molecular mass markers; Total, total cell proteins; 1-4, eluatefractions from immobilized metal affinity chromatography.

FIG. 4B shows a photograph of an immunoblot using rabbit antiserumraised to recombinant FauA to probe outer membrane proteins ofiron-starved B. pertussis strains. WT, wild-type B. pertussis; ΔfauA, B.pertussis fauA in-frame deletion mutant; ΔfauA/fauA+, B. pertussis ΔfauAmutant complemented using the wild type fauA gene carried on a plasmid.

FIG. 5A is a schematic showing an expression system for production ofFim3 and Fim3-iron receptor chimeras in B. pertussis.

FIG. 5B is a schematic showing gene assembly using a thermostable ligasecycling reaction.

FIG. 6 is a schematic showing a map of B. pertussis Fim3 functionaldomains. Regions predicted to be permissive for foreign epitopeinsertion are shown in red.

FIG. 7A is a photograph of a SDS-PAGE gel showing Fim3 and two Fim3-BhuRloop domain chimeric proteins produced in E. coli. M, molecular massmarkers; Vec, vector control; Fim3, Fim3 of B. pertussis; L3, Fim3 withBhuR loop 3; L4, Fim3 with BhuR loop 4; BhuR, outer membrane proteins ofBhuR-over-producing B. pertussis cells.

FIG. 7B is a photograph of an immunoblot of a duplicate gel (as shown inFIG. 7A) probed with BhuR-specific rabbit antiserum, demonstratingreactivity with the Fim3-BhuR loop 3 and Fim3-BhuR loop 4 chimericproteins.

FIG. 8A is a schematic showing a model of multivalent Fim3-iron receptorchimeric protein filaments on the B. pertussis cell surface. Grey ovals,Fim3 fimbrial subunits; red and yellow spheres, iron receptor loopdomains displayed on the fimbrial scaffold.

FIG. 8B is a photograph of a SDS-PAGE gel showing extraction andpurification of overproduced B. pertussis Fim3 protein filaments. M,molecular mass markers; Extract, urea extract of B. pertussis cells;PEG, Fim3 precipitated from urea extract using PEG 600; Sol, Fim3filaments after solubilization of PEG 600 precipitate (Fim3, ca. 20kDal).

FIG. 9A is a map of a plasmid for production of flagellin-iron receptorchimeric proteins in E. coli, with inducible expression driven from thetac promoter under LacI repressor control control. The site for in-frameinsertion of iron receptor loop encoding DNA segments within fliC isshown.

FIG. 9B is a model of a flagellin-iron receptor chimeric proteinmonomer, showing the dispensible region of FliC that is permissive topeptide insertions or substitutions, and the polymerization domainsinvolved in self-assembly of the flagellar filament. The loop peptide isshown in red.

FIG. 9C shows a top view of proposed hybrid flagellar filaments having11 subunits per turn and with exposed iron receptor loops (filamentsdisplaying a single or multiple different iron receptor loops areshown).

FIG. 9D is a side view of proposed hybrid flagellar filaments shown inFIG. 9C.

DETAILED DESCRIPTION

The mammalian host is a very iron-poor environment; in order to multiplyand cause disease, bacterial pathogens must use their iron uptakesystems to overcome host iron restriction. Such iron uptake systemsinclude bacterial cell surface proteins (e.g., receptors) that help takeup iron-containing heme from the host or iron-loaded bacterialsiderophores. Such proteins, however, have not been previously used aspertussis vaccine antigens. The immune response to vaccination usingthese receptor proteins (or antigenic domains thereof) would targetessential bacterial processes by blocking iron uptake, thus preventingthe establishment of infection, and also by promoting clearance of theorganism. By targeting iron uptake systems that Bordetella or othergenera of bacteria requires at both early and later stages of infection,more effective bacterial clearance and protection can be achieved.

This strategy can be used to produce acellular pertussis vaccines.Acellular vaccines typically are preferred in the U.S. Therefore,polypeptides are provided herein that include at least one domain (e.g.,an extracellular domain) from a protein that is induced in response tolow iron availability or iron starvation. These proteins typically arereceptors that transport iron-bound molecules (e.g., siderophores orheme) or iron-carrying proteins, or just the iron, into the cell. Theseproteins are referred to herein as “iron receptor proteins.” Such ironreceptor proteins, or portions thereof, can be used as antigens invaccine compositions.

There are a number of different iron receptor proteins that can be usedin an acellular pertussis vaccine as described herein. Simply by way ofexample, an iron receptor protein can be a receptor for a heme protein,or a receptor for a siderophore, or a receptor for another iron sourceor nutrient that is also a TonB-dependent receptor. A heme receptorincludes, without limitation, a BhuR protein (e.g., gene locus tagBP0347); a siderophore receptor includes, without limitation, analcaligin siderophore receptor (FauA; e.g., gene locus tag BP2463) or anenterobactin siderophore receptor (BfeA; e.g., gene locus tag BP2901),or a catechol or catecholamine receptor (e.g., BfrA, BfrD, BfrE); and aTonB-dependent receptor includes, without limitation, TonB-dependentreceptors for iron as well as TonB-dependent receptors for one or morenutrients or co-factor (see, for example, Table 1).

TABLE 1 Bordetella TonB-dependent outer membrane receptors Characterizedreceptors of known substrate specificity Receptor name Locus Tag^(a)Iron Source Substrate FauA BP2463, BPP3450, BB3900 alcaligin BfeABP2901, BPP2495, BB1942 enterobactin BhuR BP0347, BPP4185, BB4655 hemeBfrA BB4761 catecholamine, catechol BfrD BP0856, BPP3376, BB3826catecholamine, catechol BfrE BP0857, BPP3375, BB3825 catecholamine,catechol Predicted receptors of unknown substrate specificity Receptorname Locus Tag^(a) BfrB BP2016, BPP2396, BB1846 BfrC BP3663, BPP0079,BB0078 BfrF BP0736, BPP3688, BB4122 BfrG BP2922, BPP1078, BB1294 BfrHBP1138, BPP3206, BB3658 BfrI BP1962, BPP2334, BB1785 BfrZ BB4744 HemCBP0456, BPP4370, BB4956 — BPP2457, BB1905 — BP3101, BPP0746, BB0832 —BP3595, BPP3984, BB4457 — BP3077, BPP0186, BB0189 — BP1760, BPP1991,BB2179 ^(a)BP, B. pertussis; BPP, B. parapertussis; BB, B.bronchiseptica

TonB-dependent receptors are bacterial outer membrane proteins that bindand transport ferric iron chelates (siderophores), heme, vitamin B₁₂, aswell as other substrates. High affinity transport of TonB-dependentreceptor substrates across the outer membrane requires a proton-motiveforce, with the energy transduced by the TonB-ExbB-ExbD inner membraneprotein complex. The structures of twelve TonB-dependent receptors havebeen solved (some alone, some with bound ligands, and some complexedwith TonB), and forty-five crystal structures now exist for comparison.All known TonB-dependent receptors have the same domain architecture,and it is likely that all TonB-dependent receptors have this generalstructure—they all have a twenty-two-stranded transmembrane beta-barrelsurrounding a globular plug domain, and a TonB-interacting domain. Thetwenty-two beta-strands that traverse back and forth across the outermembrane to form the beta-barrel are joined on the extracellular andperiplasmic aspects by loop segments. The extracellular loop segments,along with residues on the extracellular side of the plug domain, formthe specific ligand binding sites. These extracellular loops aresurface-exposed, solvent-accessible, and highly flexible, and arespecific for binding their cognate substrate. The strong conservation ofTonB-dependent receptor domain architecture allows for accurateprediction of the transmembrane beta-strands and the extracellular loopdomains using freely available protein modeling algorithms such asPRED-TMBB, I-TASSER, ROBETTA, and SPARKS-X starting with knownTonB-dependent receptor structural data as templates.

Experimental results (see, for example, FIG. 2) show that FauA is neededfor B. pertussis growth throughout the course of mouse infection, andBhuR is required for growth during late infection, when host hemesources become available due to tissue damage. Thus, elimination of bothFauA and BhuR renders B. pertussis avirulent Targeting by vaccines ofnutrient receptors that are important during different stages ofinfection provides broader protection against the bacteria not onlyattempting to colonize, but also against those already colonizing andpersisting in the respiratory tract.

One or more of the iron receptor proteins, or portions thereof,described herein for use as antigens in vaccine compositions can bedisplayed within one or more scaffold proteins. Scaffold proteinsprovide the benefit of increased antigen multivalency, which impartsenhanced immunogenity. Multivalent antigens more effectively crosslinkadjacent B-cell receptors (antigen-specific surface immunoglobulins) toinitiate the signaling cascade that induces B-cell proliferation anddifferentiation. Furthermore, the extended crosslinking confers highavidity on the antigen-B-cell interaction. Multivalent antigensdisplaying a particular receptor loop domain on a polymeric fimbrial orflagellar scaffold can be used singly as vaccine antigens, or they canbe dissociated into monomeric subunits, mixed, and reassorted toproduced multivalent antigens displaying multiple different receptorloop domains. These polyvalent antigens can be used to generate immuneresponses to multiple distinct receptor loop domains.

A scaffold protein can be one or more fimbrial (Fim) or flagellinproteins into which specific regions or domains of an antigenicpolypeptide are introduced. B. pertussis Fim2 and Fim3 proteins arealready currently used in some existing acellular vaccine formulations.For example, an antigenic polypeptide or portions thereof (e.g., a firstantigenic polypeptide or portions thereof, a second antigenicpolypeptide or portions thereof, optionally, a third antigenicpolypeptide or portions thereof, a fourth antigenic polypeptide orportions thereof, etc., etc.) can be spliced into regions of thefimbrial protein such that the fimbrial protein acts as a scaffold todisplay the antigenic polypeptide(s). Representative fimbrial proteinsinclude, without limitation, fimbrial protein X (“FimX”), fimbrialprotein 2 (“Fim2”) or fimbrial protein 3 (“Fim3”). Without limitation,the sequences of various fim genes and Fim proteins can be found, e.g.,in GenBank Accession Nos. BP1568, BP1119, BP2674, and their orthologs inother Bordetella species.

Flagellin proteins are the major subunits of bacterial flagella, andhave been used to produce vaccine antigens for many viral and bacterialagents. Flagellin proteins have broadly conserved structural features,and large insertions that replace the variable solvent-exposed region ofE. coli flagellin are remarkably well tolerated without affectingflagellar export, assembly, or function. In addition to providing highantigen valence, flagellin polymers are recognized by the innate immunesystem as a pathogen-associated molecular pattern molecule, stimulatingadaptive immunity. Representative flagellin proteins include, e.g., FliCand FlaA, and, without limitation, the sequences of various flagellingenes and flagellin proteins can be found, e.g., in GenBank AccessionNos. BP0996 and its orthologs in other Bordetella species, and b1923 andits orthologs in E. coli and Salmonella spp.

One way to accomplish this is to express one or more antigenic proteins,or portions thereof, and a scaffold protein in one or more chimericpolypeptides. Such chimeric polypeptides can be genetically constructedusing recombinant methods known in the art. Such chimeric polypeptidesresult in polymeric, multivalent antigens that are highly effectivevaccines. As discussed herein, an antigenic polypeptide or a portionthereof can be one or more of the iron receptor proteins describedherein or a portion thereof (e.g., one or more of the TonB-dependentreceptor proteins described herein, or a portion thereof).

Polypeptides are provided herein that can be used as vaccines against B.pertussis or other Bordetella species. As indicated herein, apolypeptide used in a vaccine can be an iron receptor protein or achimeric polypeptide that includes at least one scaffold protein intowhich an antigenic polypeptide has been inserted (e.g., an iron receptorprotein and/or another antigenic polypeptide). As used herein, a“purified” polypeptide is a polypeptide that has been separated orpurified from cellular components that naturally accompany it.Typically, the polypeptide is considered “purified” when it is at least70% (e.g., at least 75%, 80%, 85%, 90%, 95%, or 99%) by dry weight, freefrom the polypeptides and naturally occurring molecules with which it isnaturally associated. Since a polypeptide that is chemically synthesizedis, by nature, separated from the components that naturally accompanyit, a synthetic polypeptide is “purified.”

In addition, nucleic acids encoding such polypeptides are providedherein. As used herein, nucleic acids can include DNA and RNA, andincludes nucleic acids that contain one or more nucleotide analogs orbackbone modifications. A nucleic acid can be single stranded or doublestranded, which usually depends upon its intended use. As used herein,an “isolated” nucleic acid molecule is a nucleic acid molecule that isfree of sequences that naturally flank one or both ends of the nucleicacid in the genome of the organism from which the isolated nucleic acidmolecule is derived (e.g., a cDNA or genomic DNA fragment produced byPCR or restriction endonuclease digestion). Such an isolated nucleicacid molecule is generally introduced into a vector (e.g., a cloningvector, or an expression vector) for convenience of manipulation or togenerate a fusion nucleic acid molecule, discussed in more detail below.In addition, an isolated nucleic acid molecule can include an engineerednucleic acid molecule such as a recombinant or a synthetic nucleic acidmolecule.

Polypeptides can be purified from natural sources (e.g., a biologicalsample) by known methods such as DEAE ion exchange, gel filtration, andhydroxyapatite chromatography. A polypeptide also can be purified, forexample, by expressing a nucleic acid in an expression vector. Inaddition, a purified polypeptide can be obtained by chemical synthesis.The extent of purity of a polypeptide can be measured using anyappropriate method, e.g., column chromatography, polyacrylamide gelelectrophoresis, or HPLC analysis. Similarly, nucleic acids can beisolated using techniques routine in the art. For example, nucleic acidscan be isolated using any method including, without limitation,recombinant nucleic acid technology, and/or the polymerase chainreaction (PCR). General PCR techniques are described, for example in PCRPrimer: A Laboratory Manual, Dieffenbach & Dveksler, Eds., Cold SpringHarbor Laboratory Press, 1995. Recombinant nucleic acid techniquesinclude, for example, restriction enzyme digestion and ligation, whichcan be used to isolate a nucleic acid. Isolated nucleic acids also canbe chemically synthesized, either as a single nucleic acid molecule oras a series of oligonucleotides.

Also provided are nucleic acids and polypeptides that differ in sequencefrom the wild type sequence. Nucleic acids and polypeptides that differin sequence from the corresponding wild type sequence can have at least50% sequence identity (e.g., at least 55%, 60%, 65%, 70%, 75%, 80%, 81%,82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% sequence identity) to the corresponding wild typesequence. In calculating percent sequence identity, two sequences arealigned and the number of identical matches of nucleotides or amino acidresidues between the two sequences is determined. The number ofidentical matches is divided by the length of the aligned region (i.e.,the number of aligned nucleotides or amino acid residues) and multipliedby 100 to arrive at a percent sequence identity value. It will beappreciated that the length of the aligned region can be a portion ofone or both sequences up to the full-length size of the shortestsequence. It also will be appreciated that a single sequence can alignwith more than one other sequence and hence, can have different percentsequence identity values over each aligned region.

The alignment of two or more sequences to determine percent sequenceidentity can be performed using the computer program ClustalW anddefault parameters, which allows alignments of nucleic acid orpolypeptide sequences to be carried out across their entire length(global alignment). See, e.g., Chenna et al., 2003, Nucleic Acids Res.,31(13):3497-500. ClustalW calculates the best match between a query andone or more subject sequences, and aligns them so that identities,similarities and differences can be determined. Gaps of one or moreresidues can be inserted into a query sequence, a subject sequence, orboth, to maximize sequence alignments. For fast pairwise alignment ofnucleic acid sequences, the default parameters can be used (i.e., wordsize: 2; window size: 4; scoring method: percentage; number of topdiagonals: 4; and gap penalty: 5); for an alignment of multiple nucleicacid sequences, the following parameters can be used: gap openingpenalty: 10.0; gap extension penalty: 5.0; and weight transitions: yes.For fast pairwise alignment of polypeptide sequences, the followingparameters can be used: word size: 1; window size: 5; scoring method:percentage; number of top diagonals: 5; and gap penalty: 3. For multiplealignment of polypeptide sequences, the following parameters can beused: weight matrix: blosum; gap opening penalty: 10.0; gap extensionpenalty: 0.05; hydrophilic gaps: on; hydrophilic residues: Gly, Pro,Ser, Asn, Asp, Gln, Glu, Arg, and Lys; and residue-specific gappenalties: on. ClustalW can be run, for example, at the Baylor Collegeof Medicine Search Launcher website or at the European BioinformaticsInstitute website on the World Wide Web.

A construct, also referred to as a vector, containing a nucleic acid(e.g., a nucleic acid that encodes a polypeptide as described herein foruse in a vaccine) is provided. Constructs, including expressionconstructs, are commercially available or can be produced by recombinantDNA techniques routine in the art. A construct containing a nucleic acidcan have expression elements, and further can include sequences such asthose encoding a selectable marker (e.g., an antibiotic resistancegene). A construct containing a nucleic acid can be fused to a secondpolypeptide that, for example, can be used in purification of theencoded polypeptide (e.g., a 6× tag polypeptide, a glutathioneS-transferase (GST) polypeptide)

Expression elements include nucleic acid sequences that direct andregulate expression of nucleic acid coding sequences. One example of anexpression element is a promoter sequence. Expression elements also caninclude introns, enhancer sequences, response elements, or inducibleelements that modulate expression of a nucleic acid. Expression elementscan be of bacterial, yeast, insect, mammalian, or viral origin, andvectors can contain a combination of elements from different origins. Asused herein, operably linked means that a promoter or other expressionelement(s) are positioned in a vector relative to a nucleic acid in sucha way as to direct or regulate expression of the nucleic acid.Expression elements in a construct can be operably linked to a codingsequence in cis or in trans; expression elements that are operablylinked to a coding sequence in trans may be in-frame with the codingsequence.

Constructs as described herein can be introduced into a host cell. Asused herein, “host cell” refers to the particular cell into which aconstruct is introduced and also includes the progeny of such a cellthat carry the construct. A host cell can be any prokaryotic oreukaryotic cell. For example, nucleic acids can be expressed inbacterial cells such as E. coli, or in insect cells, yeast or mammaliancells (such as Chinese hamster ovary cells (CHO) or COS cells). Othersuitable host cells are known to those skilled in the art. Many methodsfor introducing nucleic acids into host cells, both in vivo and invitro, are well known to those skilled in the art and include, withoutlimitation, electroporation, calcium phosphate precipitation,polyethylene glycol (PEG) transformation, heat shock, lipofection,microinjection, and viral-mediated nucleic acid transfer.

A similar strategy that takes advantage of the pertussis bacterialrequirement for iron can be used to produce whole-cell pertussis (wP)vaccines. Whole-cell vaccines typically are preferred in many non-U.S.countries around the world. Similar to the strategy described above foracellular vaccines, B. pertussis grown under iron-starvation conditionscan be used in a whole-cell vaccine. Since the whole-cell pertussisvaccines currently being used are produced by growing bacterial cells iniron-rich medium, the bacteria in those vaccines will not have producedtheir iron receptors and will lack those important antigens. On theother hand, iron-starved B. pertussis produces abundant amounts of ironuptake receptors. For example, alcaligin siderophore production isevidence of iron starvation in B. pertussis. Alcaligin siderophoreproduction level can be assessed using a siderophore detection assaysuch as a chrome azurol S universal siderophore assay, or using growthstimulation assays of siderophore-deficient mutant Bordetella indicatorstrains. Iron receptor protein production levels in iron-starved B.pertussis cells can be assessed by sodium dodecyl sulfate polyacrylamidegel electrophoresis (SDS-PAGE) analysis of bacterial cell proteins,and/or by immunoblot analysis using iron receptor specific antisera. Aswith the acellular vaccines, an immune response against the iron uptakereceptors targets essential bacterial processes by blocking iron uptake,preventing a productive infection and promoting immune clearance.

Any of the polypeptides described herein (e.g., an iron receptor proteinor a chimeric polypeptide including a scaffold protein and an antigenicpolypeptide) can be used in an acellular vaccine to protect a subjectagainst infection by B. pertussis. Similarly, the iron-starved B.pertussis described herein can be used in a whole-cell vaccine toprotect a subject against infection by B. pertussis.

Vaccines are well-known in the art, and often include a pharmaceuticallyacceptable carrier. As used herein, “pharmaceutically acceptablecarrier” is intended to include any and all excipients, solvents,dispersion media, coatings, antibacterial and anti-fungal agents,isotonic and absorption delaying agents, and the like, compatible withadministration. Pharmaceutically acceptable carriers for deliveringcompounds are well known in the art. See, for example Remington: TheScience and Practice of Pharmacy, University of the Sciences inPhiladelphia, Ed., 21^(st) Edition, 2005, Lippincott Williams & Wilkins;and The Pharmacological Basis of Therapeutics, Goodman and Gilman, Eds.,12^(th) Ed., 2001, McGraw-Hill Co. The type of pharmaceuticallyacceptable carrier used in a particular formulation can depend onvarious factors, such as, for example, the physical and chemicalproperties of the compound, the route of administration, and themanufacturing procedure. Vaccines often include an adjuvant, in additionto the primary antigen, to further increase the immune response by thesubject.

The vaccines described herein (e.g., an acellular vaccine including aniron receptor protein or a chimeric polypeptide including a scaffoldprotein and an antigenic polypeptide or a whole-cell vaccine includingiron-starved B. pertussis cells) can be used in a method of vaccinatingor immunizing a subject against a B. pertussis infection (e.g., aproductive infection). Additionally or alternatively, vaccinating orimmunizing a subject can prophylactically protect the subject againstinfection by B. pertussis.

The vaccines described herein can be administered in an effective amountto a subject. Typically, an effective amount is an amount that preventsor treats a Bordetella infection in a subject without inducing anyadverse effects. The amount of polypeptide in each dose of vaccinetypically is the minimal amount that induces an immunoprotectiveresponse in a subject without significant, adverse side effects. Theparticular amount of polypeptide in a vaccine will vary depending uponthe antigenicity of the polypeptide as well as the presence of anyadjuvant. In some instances, a vaccine dose includes between about 1 μgand about 1000 μg of protein (e.g., about 1 μg to about 200 μg; about 1μg to about 50 μg; or about 1 μg to about 10 μg of protein) in a volumeof about 50 μl to about 2 ml per dose (e.g., about 100 μl to about 1.5ml; about 250 μl to about 1 ml; about 100 μl to about 500 μl; or about100 μl to about 250 μl). A whole cell vaccine dose is standardized toOpacity Units using a WHO reference preparation (IU), with one vaccinedose not exceeding 20 IU.

The vaccines described herein can be administered to any subject thatcan be infected with a Bordetella species. For example, B. pertussis andB. parapertussis infect humans, while B. bronchiseptica occasionallyinfects humans but also infects other mammals such as canines andfelines (causing kennel cough) and pigs (causing atrophic rhinitis) andB. avium infects birds (causing turkey rhinotracheitis). Alternatively,the strategy described herein can be used to make an acellular orwhole-cell vaccine from another Bordetella species. A vaccine asdescribed herein typically is formulated to be compatible with itsintended route of administration. Suitable routes of administrationinclude, without limitation, intranasal, oral, topical, pulmonary,ocular, intestinal, and parenteral administration. Routes for parenteraladministration include intravenous, intramuscular, and subcutaneousadministration, as well as intraperitoneal, intra-arterial,intra-articular, intracardiac, intracisternal, intradermal,intralesional, intraocular, intrapleural, intrathecal, intrauterine, andintraventricular administration. For example, a vaccine as describedherein can be delivered subcutaneously.

The vaccines described herein can be made using methods that are knownin the art. For example, the acellular vaccines described herein can bemade by combining any of the polypeptides described herein (e.g., aniron receptor protein or a chimeric polypeptide including a scaffoldprotein and an antigenic polypeptide) with a suitable pharmaceuticallyacceptable carrier. A polypeptide for use in a pertussis vaccine asdescribed herein can be purified, or a polypeptide for use in apertussis vaccine as described herein can be expressed from anappropriate nucleic acid (e.g., contained within a construct; containedwithin a host cell).

In addition, the whole-cellular vaccines described herein can be made bycombining iron-starved B. pertussis cells (e.g., B. pertussis cellscultured under reduced iron or iron-starvation conditions) with apharmaceutically acceptable carrier. Iron starvation can be achieved bysub-culturing B. pertussis from iron-replete culture media to culturemedia lacking iron supplementation, or by the addition of non-utilizableiron chelators to iron-replete culture medium. As discussed herein, theproduction of alcaligin siderophore or other iron receptor proteins areevidence of iron starvation in B. pertussis, and methods of evaluatingthe production levels of alcaligin siderophore or other iron receptorproteins in iron-starved B. pertussis cells are described herein and areknown in the art. It would be appreciated that the cell culture may needto be processed in one or more ways prior to being combined with thepharmaceutically acceptable carrier. For example, the cells in the cellculture can be collected and washed to remove the iron-depleted media.Additionally or alternatively, iron-starved cells can be, for example,frozen or lyophilized.

In accordance with the present invention, there may be employedconventional molecular biology, microbiology, biochemical, andrecombinant DNA techniques within the skill of the art. Such techniquesare explained fully in the literature. The invention will be furtherdescribed in the following examples, which do not limit the scope of themethods and compositions of matter described in the claims.

EXAMPLES Example 1 Production of Whole Cell Pertussis (wP) Vaccines

B. pertussis frozen stocks (in whole sheep blood, stored at −80° C.)were streaked onto Bordet Gengou agar plates and cultured at 37° C. for48 h. Plate growth was suspended in complete Stainer-Scholte liquidmedium, and used to inoculate seed cultures in complete Stainer-Scholtemedium. After 36 to 48 h growth at 37° C. in a shaking incubator,bacteria were harvested by centrifugation, and the bacterial cells werewashed at least twice using iron-free Stainer-Scholte medium. Thiswashed seed suspension was used to inoculate complete Stainer Scholtemedium (+Fe) or iron-free Stainer Scholte medium (−Fe) to an initialcell density corresponding to ˜2×10⁸ cfu/ml (˜0.1 OD₆₀₀), and grown for36 to 48 h at 37° C. in a shaking incubator. Iron starvation status iniron-free cultures was confirmed by a siderophore detection assay. B.pertussis cells were harvested by centrifugation, and washed 3 timesusing cold sterile saline solution.

Bacteria were suspended in saline to an optical density representing˜1×10⁹ cfu/ml (confirmed by plate counts), killed by heating at 65° C.for 0.5 h and adsorbed to alum (Alhydrogel®, 2%, InvivoGen) to yield +Feand −Fe wP vaccine suspensions.

Example 2 Production of Recombinant B. pertussis Iron Receptor Proteinsin E. coli

B. pertussis iron receptor genes were PCR-amplified from genomic DNAtemplates, and cloned into pBAD/His plasmid vectors (Invitrogen) forarabinose-inducible expression in E. coli. PCR primers were designed toinclude restriction site adapters for cloning, and to replace thereceptors' start codons and N-terminal signal sequences with the plasmidvector-encoded start codon and polyhistidine tag for affinitypurification of the products.

E. coli strain TOP10 (F-mcrA Δ(mrr-hsdRAIS-mcrBC) φ80lacZΔM15 ΔlacX74mpG recA1 araD139 Δ(ara-leu)7697 galE15 galK16 rpsL(Str^(R)) endA1λ⁻)(Invitrogen) carrying recombinant pBAD/His plasmids was culturedovernight on Luria-Bertani agar plates supplemented with ampicillin (200μg/ml), then sub-cultured into Luria-Bertani broth supplemented withampicillin and grown at 37° C. in a shaking incubator untilmid-exponential phase. Arabinose (20% w/v solution) was added to 0.002%final concentration to induce receptor gene expression, and the cultureswere grown for an additional 4 h. Bacterial cells were recovered bycentrifugation, washed using 1/10 culture volume STE (50 mM Tris pH 8,100 mM NaCl, 1 mM EDTA), and resuspended in 1/10 culture volume STEbefore freezing the suspension overnight at −80° C.

Thawed cell suspensions were disrupted using a French pressure cell, andthe insoluble cell fraction was recovered by ultracentrifugation. Theinsoluble material was washed using 1/10 culture volume of TE (10 mMTris, 1 mM EDTA pH 8.0), followed by washing using 1/10 culture volumeof water.

The insoluble material was then suspended in 1/10 culture volume of DB(100 mM NaH₂PO₄, 10 mM Tris, 8 M urea) at a pH 8.0, and any insolublematerial was removed by centrifugation and discarded.

The supernatant fluid containing solubilized receptor proteins wastransferred to a 50-ml conical tube containing Ni²⁺-charged ChelatingSepharose Fast-Flow beads (Amersham) equilibrated in DB (pH 8.0), andbound on a rotating platform for 1 h at room temperature. The beads werepelleted gently, and the supernatant fluid containing the unboundproteins was discarded. The beads were gently resuspended in DB (pH 6.3)to wash away loosely-bound proteins, then the beads were again pelletedgently and the supernatant fluid was discarded. After repeating the washwith DB (pH 6.3), the slurry was transferred to a disposable columnfitted with a glass fiber support. The column bed was eluted 4 times,each time using ½ bed volume of DB (pH 5.9), and the eluates werecollected separately. Elution was repeated, except using DB (pH 4.5),and, again, the eluates were collected separately. Eluate fractions wereanalyzed by SDS-PAGE for the presence of the receptor proteins. Peakfractions were pooled and the receptor proteins were precipitated by theaddition of an equal volume of acetone at −20° C. After 30 min at −20°C., the protein precipitate was recovered by centrifugation, brieflyair-dried, then dissolved in TE containing 8 M urea. After removing anyinsoluble material by centrifugation, the cleared protein solution wasrapidly diluted into 10 volumes of 10 mM CHAPS in TE to renature theprotein. The protein solution was cleared by centrifugation or using acentrifugal filter unit with a 0.22 μm filter. Soluble receptor proteinconcentration was determined using the Bradford assay.

Example 3 Construction of Recombinant Plasmids Encoding B. pertussisFimbriae-Iron Receptor Protein Chimeras

The fim3 coding sequences with stop codon, and the fhaB promoter regionwere each PCR amplified using B. pertussis genomic DNA template and5′-phosphorylated oligonucleotide primers. The resulting products werecombined with SmaI-linearized plasmid vector pBBR1MCS-5 by the ligasecycling reaction using bridging oligonucleotides to yield recombinantplasmid pBB5IP(fhaB-fim3). This plasmid places fim3 expression under thecontrol of the strong, well-characterized Bvg-dependent fhaB filamentoushemagglutinin promoter, thus circumventing the negative influence of thefim3 downstream repressive element on fim3 expression, and the polyCtract involved in fim3 phase variation. The plasmid construct wasverified by nucleotide sequencing, and SDS-PAGE analysis confirmed thatB. pertussis carrying plasmid pBB5IPfhaB-fim3 overproduces the Fim3protein. Sub-regions of B. pertussis iron receptor genes encoding knownor predicted extracellular loop domains of the receptor proteins werePCR-amplified using oligonucleotide primers with fim3 adapters. Theproducts were recombined with fim3 by PCR megaprimer cloning atfim3sites predicted to be permissive for foreign peptide insertion withoutinterference with fimbrial assembly and maturation in B. pertussis.Plasmids encoding Fim3-iron receptor chimeric proteins were confirmed bynucleotide sequencing, then transferred to B. pertussis by mating.

TABLE 2 FauA extracellular loop domains Extra SEQ cellular Amino ID loopacids FauA amino acid residues NO  1 205-209 GSWDY  1  2 235-250SQGDSYVHFLDTRRRT  2  3 274-312 NHSNGFGSGFPLFYSDGSRTDFNRSVANNAPWARQDTEA 3  4 336-375 TDGRYLMKHVYRGGYPDRHTGIIAAPPAFSNYDGNLDRDD  4  5 398-448MSIDNHSDIQRYAMVGPAPAIGSFFDWRRAHIQEPSWADTLSPADDVRTKQ  5  6 472-492SDWKTKQMYFGSRREYRIKNQ  6  7 518-537 QPQNARDTSGGILPPIKSKS  7  8 558-593FQTRQDNLAQVIPGSSIPGFPNMQASRAASGAKVEG  8  9 615-633 FTTKDASGNPINTNHPRSL 9 10 658-679 QSRMYQAAASPRGNVEVEQDSY 10 11 701-711 NNLFDKKYYDQ 11

TABLE 3 BhuR extracellular loop domains Extra SEQ cellular Amino ID loopacids BhuR amino acid residues NO:  1 274-316HDDTSWLLQAGTRNGHDLDNRADTGGYGSKRSQPSPEDYAQNN 12  2 338-367FKRRADLDQMYQQGAGTSYQYGANRTHEET 13  3 397-442QRLRLDSSQDARRTRDGRAYARPGDPYFYGYPSGPYGRSNSIQESI 14  4 468-515YGNRTEQYSDGYDNCPAIPPGTPAPMGPRLCDMLHTNQADMPRVKGSQ 15  5 542-569YEQKPQQGGGYQNNPNAGALPPSSSGGR 16  6 595-627RAPSATELYTNYGGPGTYLRVGNPSLKPETSKG 17  7 648-660 NRYQNFIDKNVPL 18  8680-690 TGLANRARVRI 19  9 714-730 AVGKDENTGQHLNSVPP 20 10 755-778RRDDVQYPEASASARYADFQAPGY 21 11 806-836 DKKYWEAINVPTAGAIAIPRPLDWYNEPGRS22

TABLE 4PCR primers for production of DNA segments for ligase cycling assembly forconstruction of fim3-expressing plasmid pBB5/PfhaB-fim3 SEQ SEQ ProductTarget Forward ID Reverse ID size region primer NO primer NO: (bp) fhaBTACGTGCCGGACAGGGTTT 23 ATTCCGACCAGCGAAGTGAAG 24 195 promoter fim3ATGTCCAAGTTTTCATACCCTGC 25 TCAGGGGTAGACGACGGAAA 26 615 CDS

TABLE 5Bridging oligonucleotides for ligase cycling assembly with SmaI-cutplasmid vector pBBR1MCS-5 for construction of pBB5/PfhaB-fim3 SEQ IDVector Sequence NO: pBBR1MCS-5GCTCTAGAACTAGTGGATCCCCCTACGTGCCGGACAGGGTTT 27 vector/fhaB promoterfhaB promoter/ CTTCACTTCGCTGGTCGGAATATGTCCAAGTTTTCATACCCTGC 28 fim3 CDSfim3/ TTTCCGTCGTCTACCCCTGAGGGCTGCAGGAATTCGATATCA 29 pBBR1MCS-5 vector

TABLE 6 Fim3 regions predicted to be permissive for iron receptorloop domain insertion Predicted Fim3 Fim amino SEQ ID Amino acidNucleotide permissive site acid residues NO: residues positions Site 1PFDIKLKECPQALGAL 30  76-91 226-273 Site 2 SATKAKGVEFRLA 31 128-140382-420

TABLE 7PCR primers for amplification of BhuR extracellular loop domains 3 and 4,and insertion at Fim3 permissive sites 1 and 2 by the PCR megaprimermethod Fim3 SEQ SEQ BhuR insertion ID ID loop site Forward primer NOReverse primer NO 3 1 CCCTTCGACATCAAGCTGAAGCTGGAC 32 GGGCTCGAAATACAGCTTG33 AGCTCGCAGGAC AGCGATTCCTGGATCGAGT TGCT 3 2 GGCAACCTGAGCACCGTGTCGCTGGAC34 GATGTGCTGGCCGTTGAGG 35 AGCTCGCAGGAC TTCGATTCCTGGATCGAGT TGCT 4 1CCCTTCGACATCAAGCTGAAGGAGCAG 36 GGGCTCGAAATACAGCTTG 37 TACTCGGACGGCTAAGCTGGCTGCCCTTGACCC G 4 2 GGCAACCTGAGCACCGTGTCGGAGCAG 38GATGTGCTGGCCGTTGAGG 39 TACTCGGACGGCTA TTCTGGCTGCCCTTGACCC G

TABLE 8 B. pertussis Tohama I fhaB promoter region DNAsequence (SEQ ID NO: 40):TACGTGCCGGACAGGGTTTGATGGTTTGACTAAGAAATTTCCTACAAGTCTTGTATAAATATCCATTGATGGACGGGATCATTACTGACTGACGAAGTGCTGAGGTTTATCCAGACTATGGCACTGGATTTCAAAACCTAAAACGAGCAGGCCGATAACGGATTCTGCCGATTACTTCACTTCGCTGGTCGGAAT

TABLE 9 B. pertussis Tohama I fim3 DNA sequence (SEQ ID NO: 41):ATGTCCAAGTTTTCATACCCTGCCTTGCGCGCCGCGCTTATCCTTGCCGCCTCGCCCGTACTGCCAGCGCTGGCCAACGACGGCACCATCGTCATCACCGGCAGCATCTCCGACCAGACCTGCGTCATCGAAGAGCCCAGCACCCTCAACCATATCAAGGTCGTGCAACTGCCCAAGATTTCCAAGAACGCGCTCAGGAACGACGGCGACACCGCCGGCGCCACGCCCTTCGACATCAAGCTGAAGGAATGCCCCCAGGCGCTGGGCGCGCTCAAGCTGTATTTCGAGCCCGGCATCACCACCAACTACGACACGGGCGATCTGATTGCCTACAAGCAGACCTACAACGCATCCGGCAACGGCAACCTGAGCACCGTGTCGTCCGCCACCAAGGCCAAGGGCGTGGAGTTCCGCCTGGCCAACCTCAACGGCCAGCACATCCGCATGGGCACGGACAAAACCACGCAAGCCGCGCAAACCTTTACCGGCAAGGTCACCAATGGCAGCAAGAGCTACACCCTGCGCTATCTCGCCTCGTACGTGAAGAAACCCAAGGAAGATGTCGACGCGGCCCAGATCACCAGCTACGTCGGCTTTTCC GTCGTCTACCCCTGA

TABLE 10 B. pertussis Tohama I Fim3 amino acid sequence (SEQ ID NO:42):MSKFSYPALRAALILAASPVLPALANDGTIVITGSISDQTCVIEEPSTLNHIKVVQLPKISKNALRNDGDTAGATPFDIKLKECPQALGALKLYFEPGITTNYDTGDLIAYKQTYNASGNGNLSTVSSATKAKGVEFRLANLNGQHIRMGTDKTTQAAQTFTGKVTNGSKSYTLRYLASYVKKPKEDVDAAQITSYVGFS VVYP

TABLE 11Extracellular loop domains of three other Bordetella TonB-dependentreceptor proteins Extra SEQ cellular Amino ID HemC loop acidsAmino acid residues NO  1 100-122 DTEDVKIVLDGAPKGFEKYRQGS 43   2 156-231DTKDAADLLPPGARFGALAKYGRHSNDGQDIYSVAL 44YGRTRADGADGLLYANRRDGGDLRRPDGTRFAYSRNNQRS  3 253-290SNAAGWQPFAAKRDDLPAPSQADIDRYGLTEAWRRKLV 45  4 320-418ARSDTRQRDRRSSRASQSAFLGTLGNKSWVDYRDDR 46FDLSNESHVALGTAEHVLLAGLRWHRHRRDTLMYYP PGRGEPDYNHGYFQPHYMPSGTQTVRS  5441-458 VANTGRPNDAPRYNNPAP 47  6 481-485 KAARG 48  7 510-526AKSNVSGSSRALRPERI 49  8 554-595FRNRGKHEIFQRRGVACRGQAEGGAASDCPKPLSNYRNLPGY 50  9 624-643RDASPRDPWGPRTWIAEIPP 51 10 668-693 VRRQDRSPTDGDPLAGYWALPKTAGY 52 11717-738 DNLFNRPYHPYLGEAVSGTGRN 53 Extra cellular Amino BfeA loop acidsAmino acid residues  1 191-203 YTNQPEDSREGNT 54  2 227-259NKTNPDARDINAGHANTSDNGNPSTAGREGVIN 55  3 285-317QGNLFAGDTMNNANSDFSDSLYGKETNAMYREN 56  4 340-369TRNARQREGLAGGPEGAPTAGGYDTARLKN 57  5 395-430LRESLEDPAGTRQTYTGGAIGGTAPADRDPKSRQTS 58  6 456-462 NSEFGSN 59  7 488-529KAPNLYQSNPNYLLYSRGNGCLASQTNTNGCYLVGNEDLSPE 60  8 552-589FRNDYRNKIVAGTDVQYRLANGARVLQWTNSGKAVVEG 61  9 615-628 KEKATGEPLSVIPE 6210 652-675 YGKQEGPSTNVRTGVELNGDGRQT 63 11 705-728DKQLYREGNASSAGAATYNEPGRA 64 Extra cellular Amino BP3077 loop acidsAmino acid residues  1 ... ...  2 227-251 LKRRSSDYRVPDWPDGKLAGSYSES 65 3 274-339 LESKYGLPGHNHEYEGCHPHGSHLHCGGHDDHGHGH 66DEHEEGEAEHDHGHEHGAGDVPYVKLRSNR  4 363-383 TDYRHDEIEGGQLGTRFQNRG 67  5409-427 SDFRATGEEAFLPRSKTRA 68  6 452-467 QRVSPQSGAPASRTAG 69  7 493-524RLPSAQELYADGVHLATNTYEIGDPGLDRETS 70  8 547-576NRVKNYIYANTLDRYEDFRLIEYTQRDAEF 71  9 600-616 VRGRLTGGGGNLPRIPA 72 10639-657 VYRQDDIAAYESSTPGYDM 73 11 680-704 NNLLNKLAFNHASFISTVAPLPGRS 74

Example 4 Purification of Fim3-Iron Receptor Chimeric Proteins Producedin B. pertussis

Fimbriae were extracted from B. pertussis cells recovered fromStainer-Scholte liquid cultures by incubation in a solution of 4 M ureain phosphate-buffered saline at 60° C. for 30 min with gentle agitation,followed by precipitation with 4% PEG 600. In addition, the fim3-ironreceptor encoding insert DNA fragments of pBB5IPfhaB-fim3 derivativeswere sub-cloned into plasmid vector pET-3a for inducible T7promoter-directed expression and high-level production of chimericproteins in E. coli.

Example 5 Construction of Recombinant Plasmids Encoding B. pertussisIron Receptor-Flagellin Protein Chimeras

Polyvalent iron receptor antigens are produced using the FliC protein asa display scaffold. Individual B. pertussis receptor loop domain codingsequences are spliced in-frame into the dispensable region of fliC borneon a ColE1 plasmid (a pBR322 derivative). Construction of FliC-loopdomain chimeras use PCR-generated loop-encoding DNA segments asmegaprimers. Expression is driven from the tac promoter under LacIcontrol, and the product is exported and assembled on the bacterialsurface as flagella. Flagella (5-10% total cell protein) are easilypurified after mechanical shearing from the bacterium and dissociationinto monomers at reduced pH. Purified FliC monomers readily polymerizeinto filaments (ca. 20,000-30,000 monomers) in physiological pH solutionby seeding with small flagellar fragments. Different FliC-receptor loopfusion protein monomers are produced in E. coli and purified from itsflagella, then combined and assembled in vitro to produce polyvalentvaccine antigen polymers displaying multiple different iron receptorloop domains.

Example 6 Results

FIG. 1 are exemplary photographs showing that serum fromculture-positive human donors (“pertussis patient serum”) reacts withBordetella iron source receptors. Sera were from B. pertussisculture-positive children (aged 6 to 17 years old, previouslyvaccinated, n=12) and a control group of normal adult donors not knownto have been infected with B. pertussis (n=10).

FIG. 2 shows results of mouse infection studies that evidence theimportance of alcaligin siderophore and heme iron utilization systems toin vivo fitness of B. pertussis at different stages of infection. FIG. 2shows that a mutant B. pertussis strain lacking the FauA and BhuRreceptors was severely attenuated in the mouse pertussis model.

FIG. 4 are photographs showing the production of thepolyhistidine-tagged iron receptor proteins in E. coli and purificationby metal affinity chromatography. FauA is shown simply as an example.Note that the FauA-specific antiserum shows negligible cross-reactivitywith other iron receptor proteins. Similar results were obtained for theBhuR heme receptor protein, demonstrating that the receptor proteins areimmunogenic.

The plasmid construct shown in FIG. 5A directs expression of Fim3 fromthe strong Bvg-dependent fhaB promoter, thus eliminating the influenceof the downstream repressive element in the Fim3 initial transcribedregion and the Fim3 promoter poly(C)-tract effecting phase-variantexpression. B. pertussis expresses the accessory genes necessary forfimbrial assembly and export.

In addition, two different BhuR receptor protein extracellular loopdomains were each spliced into site 1 within the Fim3 fimbrial protein(FIG. 6) and the chimeric products were overproduced in E. coli. FIG. 7shows that the FIG. 3-loop chimeric proteins (L3, L4) were stronglyreactive with rabbit antisera raised to the purified recombinant BhuRreceptor protein. This result indicates that immunization using achimeric protein as described herein may elicit an anti-iron receptorimmune response in a subject that can block colonization and/or enhanceimmune recognition and clearance of the B. pertussis.

It is to be understood that, while the methods and compositions ofmatter have been described herein in conjunction with a number ofdifferent aspects, the foregoing description of the various aspects isintended to illustrate and not limit the scope of the methods andcompositions of matter. Other aspects, advantages, and modifications arewithin the scope of the following claims.

Disclosed are methods and compositions that can be used for, can be usedin conjunction with, can be used in preparation for, or are products ofthe disclosed methods and compositions. These and other materials aredisclosed herein, and it is understood that combinations, subsets,interactions, groups, etc. of these methods and compositions aredisclosed. That is, while specific reference to each various individualand collective combinations and permutations of these compositions andmethods may not be explicitly disclosed, each is specificallycontemplated and described herein. For example, if a particularcomposition of matter or a particular method is disclosed and discussedand a number of compositions or methods are discussed, each and everycombination and permutation of the compositions and the methods arespecifically contemplated unless specifically indicated to the contrary.Likewise, any subset or combination of these is also specificallycontemplated and disclosed.

What is claimed is:
 1. A chimeric polypeptide comprising at least oneantigenic polypeptide and a scaffold protein.
 2. The chimericpolypeptide of claim 1, wherein the at least one antigenic polypeptidecomprises an iron receptor protein or an antigenic portion thereof. 3.The chimeric polypeptide of claim 2, wherein the antigenic portion of aniron receptor protein comprises at least one extracellular domain. 4.The chimeric polypeptide of claim 2, wherein the iron receptor proteinis a TonB-dependent receptor protein or an antigenic portion thereof. 5.The chimeric polypeptide of claim 4, wherein the TonB-dependent receptorprotein is a ferric enterobactin siderophore (BfeA) receptor protein. 6.The chimeric polypeptide of claim 2, wherein the iron receptor proteinis a hemin or hemoprotein receptor or an antigenic portion thereof. 7.The chimeric polypeptide of claim 6, wherein the hemin or hemoproteinreceptor is a BhuR protein.
 8. The chimeric polypeptide of claim 2,wherein the iron receptor protein is a siderophore receptor or anantigenic portion thereof.
 9. The chimeric polypeptide of claim 8,wherein the siderophore receptor is an alcaligin siderophore receptor(FauA).
 10. The chimeric polypeptide of claim 1, wherein the scaffoldprotein is a fimbrial protein or a flagellin protein.
 11. The chimericpolypeptide of claim 10, wherein the fimbrial protein is a fimbrial 2protein or fimbrial 3 protein.
 12. The chimeric polypeptide of claim 10,wherein the flagellin protein is a flagellin subunit protein.
 13. Anucleic acid molecule encoding the polypeptide of claim
 1. 14. Aconstruct comprising the nucleic acid molecule of claim
 13. 15. A hostcell comprising the nucleic acid molecule of claim
 13. 16. A host cellcomprising the construct of claim
 14. 17. An acellular B. pertussisvaccine for protecting a subject against infection by B. pertussis, B.parapertussis, B. bronchiseptica and/or B. avium, comprising thechimeric polypeptide of claim 1 and a pharmaceutically acceptablecarrier.
 18. The acellular vaccine of claim 17, further comprising anadjuvant.
 19. A whole cell B. pertussis vaccine for protecting a subjectagainst infection by B. pertussis, B. parapertussis, B. bronchisepticaand/or B. avium, comprising a composition of B. pertussis grown underiron-starvation conditions and a pharmaceutically acceptable carrier.20. The whole cell vaccine of claim 19, further comprising an adjuvant.21. A method of vaccinating a subject against B. pertussis, comprising:administering, to a subject, the polypeptide of claim 1 or the vaccineof any of claim 17, 18, 19, or
 20. 22. The method of claim 21, whereinthe subject is a human, a canine, a pig, a rabbit, a cat, or a bird. 23.A method of making an acellular B. pertussis vaccine, comprising:providing the polypeptide of claim 1; or expressing the nucleic acidmolecule of claim 13 or the construct of claim 14; or culturing the hostcell of claim 15 or 16; and combining the polypeptide produced therefromwith a pharmaceutically acceptable carrier.
 24. The method of claim 23,further comprising adding an adjuvant.
 25. A method of making awhole-cell B. pertussis vaccine, comprising: culturing B. pertussisunder iron-starvation conditions; and processing the B. pertussis grownunder iron-starvation conditions into a whole-cell vaccine.
 26. Themethod of claim 25, further comprising adding an adjuvant.